Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Summary

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

Abstract

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Introduction

High molecular weight BDA (10,000 molecular weight), a highly sensitive tracer, has been used for tracing neural pathways in the central nervous system for over 20 years¹. Although the use of the BDA is a common neural tract tracing technique, the quality of BDA labeling can be affected in animals by various factors^1,2,3. Our recent study indicated that the optimal structure of BDA labeling is not only associated with a proper post-injection survival time, but also correlated with the staining method⁴. Until now, conventional av....

Protocol

This study was approved by the ethics committee at the China Academy of Chinese Medical Sciences (reference number 20160014). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996). Four adult male rats (weight 250-280 g) were used in this study. All animals were housed in a 12 h light/dark cycle with controlled temperature and humidity, and allowed free access to food and water. The instruments and materials used in the present study were showed in Figure 1.  Before the surgery, all instruments, such as ....

Results

Survival of 10 days post injection of BDA into the VPM was sufficient for producing intense neural labeling on the corresponding cortical areas ipsilateral to the injection side (Figure 2). Both conventional ABC and fluorescent staining procedures for BDA revealed the similar pattern of neural labeling on the S1, including anterogradely labeled thalamocortical axons and retrogradely labeled corticothalamic neurons (Figure 2C

Discussion

Selecting a proper tracer is a critical step for a successful neural tracing experiment. In the family of BDA, high molecular weight BDA (10,000 molecular weight) was recommended to be preferentially transported through the anterograde neural pathway in contrast to low molecular weight BDA (3,000 molecular weight)^2,3,11,12,13. However, many studies also sugges.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biotinylated dextran amine (BDA)	Molecular Probes	D1956	10,000 molecular weight
Streptavidin-Alexa Fluor 594	Molecular Probes	S32356	Protect from light
500/525 green fluorescent Nissl stain	Molecular Probes	N21480	Protect from light
Brain stereotaxis instrument	Narishige	SR-50
Freezing microtome	Thermo	Microm International GmbH
Confocal imaging	Olympus	FV1200
system
Micro Drill	Saeyang Microtech	Marathon-N7
Sprague Dawley	Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences	SCKX (JUN) 2012-004
Vectastain ABC Kit	Vector Laboratories	PK-4000
superfrost plus microscope slides	Thermo	#4951PLUS-001	25x75x1mm
Photoshop and Illustration	Adobe	CS5